Abstract Aneuploidy, a cellular state of having an abnormal number of chromosomes, is a hallmark of cancer. The degree of aneuploidy significantly correlates with tumor aggressiveness and poor clinical prognosis. Therefore, studying the cellular processes affected by aneuploidy can improve our understanding of the role of aneuploidy in tumor biology. Our preliminary results show that de novo synthesis of sphingolipids is increased by aneuploidy; we therefore propose to identify the molecular mechanisms underlying this effect. Targeting the synthesis of sphingolipids holds great potential as an anti-cancer strategy that could be used either alone or in combination with existing therapies. Despite the existence of a large body of literature providing strong evidence for the misregulation of sphingolipid metabolism in human diseases, including several types of cancers, the molecular mechanisms that lead to this misregulation are poorly understood. The focus of this proposal is to unravel the molecular mechanisms that regulate de novo synthesis of sphingolipids and to decipher how these mechanisms are affected in aneuploid cells. To this end, we propose to: (1) Determine how aneuploidy increases sphingolipid biosynthesis. Our preliminary data supports the hypothesis that aneuploid cells rely on the increased activity of serine palmitoyltransferase, the enzyme that controls the first and irreversible step of sphingolipid synthesis, to proliferate. Therefore, we will investigate the function of signaling pathways that regulate serine palmitoyltransferase in aneuploidy. In addition, because serine serves as a precursor for sphingolipids, we will investigate whether aneuploidy leads to increased serine utilization. To that end, we plan to quantify serine metabolic flux through the sphingolipid pathway in aneuploid cells. Our studies will provide novel insights into how sphingolipid synthesis is affected in response to aneuploidy. (2) Determine how sphingolipid levels control the fitness of aneuploid cells. Our preliminary results show that mutations in four different genes that increase the levels of sphingosine and lower those of ceramide, improve the fitness of aneuploid cells. Therefore, we will determine, in aneuploid cells, the function of Pkh1/2 kinases and Cdc55 phosphatase because these signaling molecules are known to act downstream of sphingolipids and regulate the cell cycle and responses to stress. In addition, we will determine specific cellular pathways and processes that play an important role in overcoming the detrimental effects of aneuploidy. Gene expression, proteome content, and phenotypic analyses in combination with genetic approaches will be used to accomplish this aim. Altogether, our studies will contribute to a better understanding of the physiological role of sphingolipids in controlling the fitness of aneuploid cells. Determining the mechanisms that control the fitness of aneuploid cells can be exploited to target aneuploid cancer cells and to ameliorate the deleterious effects of aneuploidy in Down syndrome or neurodegenerative diseases.